The present invention relates to a method for measuring activity of osteoclast-derived acid phosphatase which is specific for bone metabolism. According to the present invention, the measurement of osteoclast-derived acid phosphatase activity can be carried out easily and specifically. And, the method is very useful measurement for clinical tests in the field of medical treatment and clinical assessment.
Acid phosphatases are enzymes having acidic optimal pH, and hydrolyzing organic mono-phosphoric esters. Various kinds of the enzymes such as prostate-derived acid phosphatase, osteoclast-derived acid phosphatase, erythrocyte-derived acid phosphatase and platelet-derived acid phosphatase have been known and the enzymes can be separated into 6 bands of 0 to 5 by electrophoresis. Tartrate-resistant acid phosphatases in serum whose enzyme activities are not inhibited by addition of a tartrate are present at Band 5 and almost all of them are thought to be osteoclast-derived acid phosphatase. Then, their measurement is thought to be useful as an indicator for evaluating functions of osteoclasts and is expected to be a marker for bone resorption [(Hone-taisha (Bone Metabolism) Marker, Hitoo, Fukunaga, et al., Medical Review, 1995).
Generally, for measuring tartrate-resistant acid phosphatases in a sample by enzyme activity measurement, their activity is determined by colorimetry of a product (such as alcohol and phenols) produced by an enzyme reaction using a phosphoric ester as a synthetic substrate in the presence of a tartrate. Upon the reaction, the tartrate inhibits mainly prostate-derived acid phosphatase which co-exists in the sample. Therefore, tartrate-resistant acid phosphatase activity is determined as an indicator of osteoclast-derived acid phosphatase activity by measuring the remaining acid phosphatase activity using the substrate. In a method known to be more improved and specific measurement of osteoclast-derived acid phosphatase activity, serum is diluted 5 times, the resultant solution is incubated at 37xc2x0 C. for 1 hour, and then the remaining tartrate-resistant acid phosphatase activity is measured using p-nitrophenyl phosphoric acid as the substrate in the presence of a tartrate (Nichi-dai ishi, Vol. 49, 904-911, 1990: Clin. Chem., Vol. 3, 458-462, 1987).
The above methods, wherein tartrate-resistant acid phosphatase activity is measured as an indicator of osteoclast-derived acid phosphatase, have problems in specificity, sensitivity, complexity of measurement and measuring time. In a sample, there are erythrocyte-derived acid phosphatase and platelet-derived acid phosphatase as tartrate-resistant acid phosphatase in addition to osteoclast-derived acid phosphatase. That is, when hemolysis occurs by collection of a sample, erythrocyte-derived acid phosphatase is contained in the sample. Also, when serum is used as a sample, platelet-derived acid phosphatase is contained in the sample by destruction of platelets during blood coagulation in preparation of the serum. Thus, it cannot be said that the above measurement of tartrate-resistant acid phosphatase activity determines osteoclast-derived acid phosphatase activity specifically.
For a method for measuring osteoclast-derived acid phosphatase without influence of erythrocyte and platelet-derived acid phosphatases, the present inventors previously found that there is a difference in resistance to a fluoride between erythrocyte- and platelet-derived acid phosphatases and osteoclast-derived acid phosphatase, and reported a method for determination of osteoclast-derived acid phosphatase activity by measuring both tartrate-resistant acid phosphatase activity and tartrate-resistant fluoride-resistant acid phosphatase activity, and then calculating the difference between them (JP 10-337198 A).
Tartrate-resistant acid phosphatases in serum are located at a fifth band from the starting point by electrophoresis and are called Band 5 tartrate-resistant acid phosphatase (hereinafter, sometimes, referred to as xe2x80x9cacid phosphatase located at Band 5xe2x80x9d). Band 5 tartrate-resistant acid phosphatase can be separated into a component located at Band 5a and has many binding glycoproteins and another component located at Band 5b and has less binding glycoproteins (hereinafter, sometimes, referred to as xe2x80x9cBand 5a tartrate-resistant acid phosphatasexe2x80x9d or xe2x80x9cacid phosphatase located at Band 5axe2x80x9d and xe2x80x9cBand 5b tartrate-resistant acid phosphatasexe2x80x9d or xe2x80x9cacid phosphatase located at Band 5bxe2x80x9d, respectively.). Production processes and functional mechanisms in a living body of Band 5a and Band 5b tartrate-resistant acid phosphatases are not yet elucidated. However, Band 5b tartrate-resistant acid phosphatase varies with bone metabolism, while Band 5a tartrate-resistant acid phosphatase dose not vary. In view of this, Band 5b tartrate-resistant acid phosphatase is considered to be very concerned with bone metabolism and to be osteoclast-derived acid phosphatase (Lam, K. W., Lai, L., and Yam, L. T., Clin. Chem. , 1978, 24, 309-312). Therefor, a simple method for specific measurement of Band 5b tartrate-resistant acid phosphatase has been desired.
In the method for measuring osteoclast-derived acid phosphatase activity reported in the above JP 10-337198 A, tartrate-resistant fluoride-sensitive acid phosphatase is regarded as osteoclast-derived acid phosphatase. Then, the method cannot differentiate acid phosphatase activity of Band 5a acid phosphatase having many binding glycoproteins from that of Band 5b acid phosphatase having less binding glycoproteins, and cannot measure specifically the activity of acid phosphatase which is specific for bone metabolism.
In view of these circumstances, an object of the present invention is to provide a simple, economical and clinically available method for specific measurement of activity of osteoclast-derived acid phosphatase which is specific for bone metabolism and located at Band 5b, and a kit for using in the method.
The present inventors have studied intensively to solve the above problems in the prior art. As a result, it has been found that the activities between Band 5a and Band 5b tartrate-resistant acid phosphatases can be differentiated from each other by treating tartrate-resistant acid phosphatase with an acid mucopolysaccharide. Thus, the present invention has been completed.
That is, according to the present invention, there is provided a method for measuring activity of osteoclast-derived acid phosphatase located at Band 5b of Band 5 in polyacrylamide gel electrophoresis of a sample which comprises using an inhibitor to acid phosphatases located at Band 5 other than Band 5b. 
More particular, the present invention provides a method for measuring activity of osteoclast-derived acid phosphatase located at Band 5b of Band 5 in polyacrylamide gel electrophoresis of a sample which comprises:
(i) treating acid phosphatases in the sample with a substrate for the measurement of acid phosphatase activity in the presence of an inhibitor to prostate-derived acid phosphatase and an inhibitor to acid phosphatases located at Band 5 other than Band 5b to measure acid phosphatase activity (hereinafter, sometimes, referred to as xe2x80x9cmeasurement 1xe2x80x9d);
(ii) independently of (i), treating acid phosphatases in the sample with a substrate for the measurement of acid phosphatase activity in the presence of an inhibitor to prostate-derived acid phosphatase and an inhibitor to acid phosphatases located at Band 5 to measure acid phosphatase activity (hereinafter, sometimes, referred to as xe2x80x9cmeasurement 2xe2x80x9d); and then,
(iii) subtracting the activity of the above (ii) from the activity of the above (i) to obtain the activity of osteoclast-derived acid phosphatase located at Band 5b. 
The present invention also provides a composition and a kit for using in these methods.